Summary Universal or 'broad-range' eubacterial polymerase chain reaction (PCR) was performed on 53 isolates from environmental water-associated sites in a haematology unit (N=22), and the outer surfaces of cleaning lotion containers sited throughout a tertiary referral hospital (N=31). 16 S rDNA PCR was performed using two sets of universal primers, including the novel reverse primer, XB4, to generate a composite amplicon of 1068 bp, which was sequenced to obtain each isolate's identity. Sequence analysis was able to identify 51 isolates. Most (75% from the haematology unit and 81% from cleaner containers) were Gram-positive. Nine different genera were identified from the haematology unit and 13 from the cleaning lotion containers. This study provides the first reports of Terrabacter spp. And Brachybacterium paraconglomeratum isolated from a hospital environment. As unusual and difficult-to-identify environmental organisms are unlikely to be clinically significant, and molecular identification is costly and labourintensive, we recommend that molecular methods are only used as an adjunct to first-line phenotypic identification schemes where a definitive identification is required. Where molecular identification methods are justified, partial 16 S rDNA PCR and sequencing employing the novel universal primer XB4, is a valuable and reliable technique.

Edible seaweed (Palmaria palmata) has traditionally been consumed rawafter harvesting from coastal shorelines around Northern Ireland.To date, there have been no reports examining the microbiology of this material, hence itwas the aimof this study to detect the diversity of themicro£ora found on readytoeat produce. Conventionalmicrobiological analyses of the product failed to detect any gastrointestinal pathogens and gave a mean total count of 1•3×105 cfug-1. 16S rRNA sequencing of culturable bacteria identified the Sanguibacter/Oerskovia/Cellulomonas complex, the Clavibacter/Frigoribacterium/urtobacterium complex, Enterobacter agglomerans, Erwinia herbicola, Flavobacterium spp., Micrococcus lylae, Microbacterium spp., Corynebacterium spp., and Dietza maris. A comparison of growth of isolated environmental organisms was performed to ascertain the most appropriate artificial culture media to employ for their culture in vitro. Tryptone soya broth with yeast extract (TSBYE) and brainfiheart infusion brothwith yeast extract (BHIYE) may be employed as suitable basal broth media for the laboratory culture of these organisms.This is the ¢rst preliminary report on the microbial diversity of edible seaweed and demonstrated the presence of several halophilic genera and species in fresh ready-to-eat edible seaweed from Northern Ireland. Although no gastrointestinal pathogens were cultured from this material, a larger study requiring examination of seasonal e¡ects, quality of marine water and e¡ect of drying on faecal pathogens, is required to support a functional HACCP-based approach to ensuring safety of this product.

J. E. MOORE, J. XU, B.C. MILLAR, J. COURTNEY AND J. S. ELBORN. 2003. Aims: To develop a selective agar medium to help detect and quantify Gram-negative flora in the sputum of patients with cystic fibrosis (CF). Methods and Results: A novel Gram-negative Selective Agar (GNSA) medium was developed consisting of tryptone soya broth (30g), bacteriological agar no.1 (10g), yeast extract (5g), crystal violet (2mg), nisin (48mg), novobiocin (5mg), cycloheximide (100mg), amphotericin (2mg) and double distilled water (1 l), for the selective culture of all Gram-negative flora from the sputum of patients with CF. GNSA was able to support the proliferation of all 34 Gram-negative organisms examined, including 23 species most commonly associated with CF, but was unable to support the growth of the 12 Gram-positive or seven fungal organisms examined. Sensitivity studies demonstrated that the GNSA medium was able to detect not less than 1•50×102 CFU ml-1 sputum Pseudomonas aeruginosa, 2•38×102 CFU ml)1 sputum Burkholderia cepacia genomovar IIIb and 6•70×103 CFU ml-1 sputum Stenotrophomonas maltophilia. A comparison of the microbial flora detected in the sputa of 12 adult CF patients by employment of routine bacteriological agar media and GNSA, demonstrated that GNSA was able to detect all Gram-negative organisms cultured by routine media, but had the advantage of detecting Alcaligenes xylosoxidans in two CF patients, whom had no previous history of Gram-negative infection. Conclusions: GNSA was unable to support the proliferation of any Gram-positive organism or yeast/fungi, but was successful in supporting the growth of all Gram-negative organisms challenged. Significance and Impact of the Study: Employment of this medium coupled with semi-automated technology may aid in helping to efficiently determine Gram-negative loading of respiratory secretions, particularly in response to antibiotic intervention.

A study was carried out to compare the API20C technology with polymerase chain reaction amplification and direct sequencing of the short internal transcribed spacer region 2 (ITS2) for the identification of 58 isolates of invasive candida species obtained from patients with bloodstream infections over the seven year period 1994 to 2000. Overall, there was only one disagreement between the phenotypic and genotypic identification, where the API scheme identified the isolate as C albicans but the molecular method identified it as C dubliniensis. This study demonstrated that the API20C method is useful in the identification of Candida spp isolated from blood culture and that molecular methods do not enhance identifications made using the API20C scheme. However, for correct reporting of C dubliniensis, an emerging bloodborne pathogen, it is recommended that all isolates identified as C albicans by the API20C scheme are further examined phenotypically and/or genotypically.

This study investigated the various commercially available kits and 'in-house' methods to extract DNA from Gramnegative and Gram-positive bacteria, yeast and fungal agents in commonly employed blood culture material. The main methods investigated were as follows; Qiagen QIAmp Blood kit, Roche high PCR template preparation kit, Puregene DNA extraction kit, boiling, glass beads/sonication and wash/ alkali/heat lysis. The results indicated that a simple wash/alkali/heat lysis method was the most sensitive, reproducible, simple and cost-effective extraction method. This was the only method which removed any PCR inhibitors and inherent DNA which existed in virgin BacT/Alert aerobic, anaerobic and paediatric blood culture material. Contaminating microbial DNA from Lactococcus lactis or Bacillus coagulans was identified in all batches of BacT/Alert